TIME AND PROCESS OF SPERM PENETRATION INTO HAMSTER OVA IN VIVO AND IN VITRO R. YANAGIMACHI Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts, U.S.A., and Z°°l°Sical Institute, Faculty of Science, Hokkaido University, Sapporo, Japan {Received 18th September 1965) Summary. Female golden hamsters were mated at various times before or after ovulation, and the time of sperm penetration into the ova was examined. When the females were mated several hours prior to ovula- tion, penetration of spermatozoa into the ova started about 3 hr after the commencement of ovulation or about 1\m=.\5 hr after the first ovum passed into the Fallopian tube; virtually all the ova were penetrated in the next 4 hr. In the females mated during or several hours after ovula- tion, sperm penetration occurred between 3 and 6 hr after coitus. The experiments in which spermatozoa were deposited artificially in the uteri of females shortly after ovulation, demonstrated that the sperma- tozoa recovered from the uterus of other females penetrated ova significantly faster than epididymal spermatozoa. The uterine sperma- tozoa deposited about the ova in vitro were also able to penetrate the ova more quickly than epididymal spermatozoa. The whole process of sperm penetration through the zona pellucida was observed on one occasion. This particular observation showed that: (1) the acrosome (at least, the outer acrosome membrane) of the fertilizing spermatozoon was absent before the spermatozoon started to penetrate the zona pellucida; (2) the direction of the passage of the spermatozoon was not vertical, but at an angle to the surface of the zona pellucida; (3) the time required for the sperm head to traverse the zona pellucida and the perivitelline space was 3 to 4 min and 1 to 2 sec, respectively; and (4) the head of the spermatozoon lay flat on the vitelline surface and sank into the vitellus without lively movements of the sperm flagellum. INTRODUCTION The time relationship between mating and penetration of spermatozoa into the ova has been studied in several species of laboratory animals. Austin & Braden (1954) reported that when rats were mated before ovulation, penetra¬ tion of spermatozoa into the ova did not start immediately after ovulation, but 2 to 4 hr later. A similar delay was observed in the mouse (Braden & Austin, 1954) and in the golden hamster (Austin, 1956; Strauss, 1956), where it was 3 to 5 hr and 2 hr respectively. Such an interval between ovulation and the commencement of sperm penetration was interpreted as suggesting the 359 Downloaded from Bioscientifica.com at 10/04/2021 08:07:47AM via free access 360 R. Yanagimachi necessity for physiological maturation of the ova (Austin, 1956; Strauss, 1956) or of the cumulus oophorus surrounding the ova (Braden, 1962). When females are mated before or shortly after ovulation, follicular cells surrounding the ova disperse after the ova have been penetrated by spermatozoa. This is particu¬ larly true of the mouse (Lewis & Wright, 1935), rat (Austin, 1948; Odor & Blandau, 1951) and rabbit (Moricard & Bossu, 1949), although the ability of spermatozoa to penetrate the naked ova is well established (Austin, 1951; Chang & Bedford, 1962). Evidence has also been presented to show that there is a delay between mating and sperm penetration of the ova, even when the animals are mated several hours after ovulation. This is about 2 hr in the rat (Austin & Braden, 1954) and about 4 hr in the hamster (Strauss, 1956). Since the first spermatozoon may reach the site of fertilization (ampullary portion of the Fallopian tube) within 1 hr of coitus (Blandau & Money, 1944; Austin, 1952; Yanagimachi & Chang, 1963), the interval between mating and the commencement of sperm penetration may be ascribed to the necessity for the spermatozoa to undergo physiological maturation or 'capacitation' in the female genital tract (Austin, 1952). In the mouse, the capacitation of spermatozoa seems to occur very rapidly, since sperm penetration starts as early as about 1 hr after coitus when the females are mated after ovulation (Braden & Austin, 1954). On the other hand, there is evidence that rabbit spermatozoa need 5 to 8 hr for full capacita¬ tion (Austin, 1951; Chang, 1951, 1959; Noyes, Walton & Adams, 1958). Thus, the time necessary for capacitation of spermatozoa seems to vary according to the species. Once the time relationship between mating and sperm penetration is deter¬ mined, it is not difficult to observe the process of sperm entry into the ova. Thus, Austin (1951), Austin & Braden (1956) and Blandau & Odor (1952) were able to observe various stages of sperm penetration into the living ova of the rat, mouse and hamster with the phase-contrast microscope. These workers, however, could not observe the actual process of sperm penetration through the zona pellucida. So far, only Pincus (1930) and Shettles (1953) have re¬ ported on the manner in which the spermatozoon penetrates the zona pellucida of the mammalian ovum. The purpose of the present paper is to furnish further data on the time rela¬ tionship between mating (or artificial insemination) and sperm penetration into the hamster ovum, and to give a description of the process of sperm penetration through the zona pellucida, which has been observed during the course of the study. MATERIALS AND METHODS Mating of animals Adult virgin female golden hamsters weighing 80 to 150 g were used. Under controlled light conditions (light, from 06.00 hours to 18.00 hours; darkness, from 18.00 hours to 06.00 hours), most females came into oestrus between 17.00 hours and 22.00 hours every 4th day; oestrus lasted till next morning (05.00 to 09.00 hours). Ovulation (rupture of ovarian follicles) occurred most frequently about 8 hr after the onset of oestrus (Harvey, Yanagimachi & Chang, 1961). Downloaded from Bioscientifica.com at 10/04/2021 08:07:47AM via free access Sperm penetration into hamster ova 361 Some females were mated immediately or shortly after the onset of oestrus. Other females were not allowed to mate until the time of ovulation or several hours afterwards. Each female was permitted coitus with two fertile males for about i hr. Artificial insemination Females which had been mated 4 to 5 hr previously were killed, and the mass of spermatozoa engorging the uterine horn was squeezed out and mixed thoroughly with 4 ml of Hanks' balanced salt solution in a watch glass. The sperm suspension thus prepared was used for insemination only if more than half of the spermatozoa displayed vigorous progressive movement. The density of spermatozoa in the suspension was about 25,000 to 80,000 cells/mm3. In a similar way, a suspension was prepared with spermatozoa collected from the uteri of females which were killed about \ hr after the start of coitus. The spermatozoa in these two suspensions were designated as 4 to 5 hr and \ hr uterine spermatozoa, respectively. A suspension of epididymal spermatozoa was prepared by placing one cauda epididymidis of a male in 4 ml of Hanks' solution, mincing it up with scissors, stirring thoroughly and then letting the tissue debris precipitate. The density of spermatozoa in the suspension was about 40,000 to 100,000 cells/mm3, and these usually showed high motility. The females to be inseminated were anaesthetized with ether about 10 hr after the onset of oestrus (i.e. about 2 hr after the peak of ovulation) and the uteri were exposed through a mid-ventral incision. About 0-2 ml of the sperm suspension was injected into the lumen of each uterine horn by means of a No. 25 gauge needle attached to a tuberculin syringe. Insemination of ova in vitro Unfertilized ova were recovered from the Fallopian tubes between 10 and 13 hr after the onset of oestrus. The method of insemination of the ova in vitro has been described elsewhere (Yanagimachi & Chang, 1964). Tyrode's balanced salt solution was used to suspend spermatozoa for insemination. The density of spermatozoa in the suspension was 4000 to 8000 cells/mm3 in most cases.* Examination of ova Between 2 and 14 hr after mating or artificial insemination, the excised Fallopian tubes were flushed with Hanks' solution (or 0-9% NaCl). If the ova were still surrounded by follicular cells, a small quantity of hyaluronidase was added to disperse them. The ova which had been inseminated in vitro were always free from the surrounding follicular cells when examined 2 to 6 hr after insemination ; the quick dispersion of follicular cells was most probably due to the action of sperm hyaluronidase. The ova, after being briefly rinsed with Hanks' solution (or 0-9% NaCl), were mounted between a slide and a coverslip supported by four dots of vaseline-paraffin mixture, pressed moderately under the coverslip and examined * In a previous paper (Yanagimachi & Chang, 1964), the density of spermatozoa in the suspension was misprinted; 4000 to 8000 cells/mm3 is correct, not 4000 to 8000 cells/ml. Downloaded from Bioscientifica.com at 10/04/2021 08:07:47AM via free access 362 R. Yanagimachi with a phase-contrast microscope for evidence of sperm penetration. An ovum was recorded as being 'penetrated' when a spermatozoon was found in the perivitelline space or in the vitelline surface, or a swelling sperm head or a developing male pronucleus was discernible in the vitellus. Observation of a spermatozoon in the act ofpenetrating the zona pellucida Thirty females were mated toward the end of oestrus (05.00 to 09.00 hours) and killed 3 to 5 hr later to remove the Fallopian tubes. After removal of the blood adhering to the tube, the tube was put at the centre of four vaseline dots on a slide and immediately covered with a drop of liquid paraffin.